Characterization of the Low Molecular Weight Human Serum Proteome

Abstract

Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome. Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome. A major goal of biomedical research is the determination of biomarkers whose measurement would effectively distinguish the onset of a defined disease state. For a biomarker (or set of biomarkers) to be clinically valuable it must not only be histologically specific but readily obtainable from the patient. While urine is widely used in diagnostic medicine, serum is potentially the most valuable specimen for biomarker elucidation (1Ardekani A.M. Liotta L.A. Petricoin 3rd, E.F. Clinical potential of proteomics in the diagnosis of ovarian cancer. Expert Rev. Mol. Diagn. 2002; 2: 312-320Google Scholar). Because serum constantly perfuses tissues, it might be expected that the onset or presence of disease may be determined by measuring the altered presence or abundance of the constituent molecular species in serum. For example, increased serum levels of prostate-specific antigen (2Grossklaus D.J. Smith J.A. Shappell S.B. Coffey C.S. Chang S.S. Cookson M.S. The free/total prostate-specific antigen ratio (%fPSA) is the best predictor of tumor involvement in the radical prostatectomy specimen among men with an elevated PSA. Urol. Oncol. 2002; 7: 195-198Google Scholar) and CA125 (3Whitehouse C. Solomon E. Current status of the molecular characterization of the ovarian cancer antigen CA125 and implications for its use in clinical screening. Gynecol. Oncol. 2003; 88: S152-S157Google Scholar) are routinely used for the detection of cancer in the prostate and ovary, respectively. Serum is attracting increasing interest in proteomics, which is currently striving to broadly characterize its protein constituents. The expectation is that the characterization of the thousands of individual serum proteins/peptides will enable the discovery of an increasing number of reliable disease biomarkers. At first glance, serum presents many beneficial attributes for proteomic investigation because it has a high protein content (i.e. 60–80 mg/ml), with many of these proteins being secreted and shed from cells and tissues (4Sasaki K. Sato K. Akiyama Y. Yanagihara K. Oka M. Yamaguchi K. Peptidomics-based approach reveals the secretion of the 29-residue COOH-terminal fragment of the putative tumor suppressor protein DMBT1 from pancreatic adenocarcinoma cell lines. Cancer Res. 2002; 62: 4894-4898Google Scholar, 5Kennedy S. The role of proteomics in toxicology: identification of biomarkers of toxicity by protein expression analysis. Biomarkers. 2002; 7: 269-290Google Scholar). The protein content of serum, however, is dominated by a handful of proteins such as albumin, transferrin, haptoglobulin, immunoglobulins, and lipoproteins (6Turner M.W. Hulme B. The Plasma Proteins: An Introduction. Pitman Medical & Scientific Publishing Co., Ltd., London1970Google Scholar). Unfortunately, serum proteins are present across an extraordinary dynamic range of concentration that is likely to span more than 10 orders of magnitude, which separates albumin from the rarest proteins now measured clinically (7Anderson N.L. Anderson N.G. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics. 2002; 1: 845-867Google Scholar). This large dynamic range exceeds the analytical capabilities of traditional proteomic methods, making the detection of lower abundance serum proteins extremely challenging. The reduction of sample complexity (e.g. to deplete the level of abundant proteins) is thus an essential first step in the analysis of the serum proteome. Affinity methods (e.g. anti-human serum albumin antibody columns, protein A/G) have been developed to remove abundant proteins such as albumin and immunoglobulins from serum prior to mass spectrometric analysis (8Sato A.K. Sexton D.J. Morganelli L.A. Cohen E.H. Wu Q.L. Conley G.P. Streltsova Z. Lee S.W. Devlin M. DeOliveira D.B. Enright J. Kent R.B. Wescott C.R. Ransohoff T.C. Ley A.C. Ladner R.C. Development of mammalian serum albumin affinity purification media by peptide phage display. Biotechnol. Prog. 2002; 18: 182-192Google Scholar, 9Adkins J.N. Varnum S.M. Auberry K.J. Moore R.J. Angell N.H. Smith R.D. Springer D.L. Pounds J.G. Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry. Mol. Cell. Proteomics. 2002; 1: 947-955Google Scholar). One of the fundamental oversights of serum protein depletion methodologies, however, is that many important low molecular weight (LMW) 1The abbreviations used are: LMW, low molecular weight; μLC, micro-capillary reverse-phase liquid chromatography; SELDI, surface-enhanced laser/desorption ionization; TOF, time-of-flight; MS, mass spectrometry; MWCO, molecular weight cutoff; MS/MS, tandem mass spectrometry; IT-MS, ion-trap mass spectrometer; CID, collision-induced dissociation; SCX, strong cation exchange. 1The abbreviations used are: LMW, low molecular weight; μLC, micro-capillary reverse-phase liquid chromatography; SELDI, surface-enhanced laser/desorption ionization; TOF, time-of-flight; MS, mass spectrometry; MWCO, molecular weight cutoff; MS/MS, tandem mass spectrometry; IT-MS, ion-trap mass spectrometer; CID, collision-induced dissociation; SCX, strong cation exchange. proteins or peptides can be concomitantly removed by this sample preparation process as well. It is well known that albumin acts as a carrier and transport protein within the blood and binds physiologically important species such as hormones, cytokines, and lipoproteins (10Burtis C.A. Ashwood E.R. Tietz Fundamentals of Clinical Chemistry. 5th Ed. W. B. Saunders Company, Philadelphia, PA2001Google Scholar), and since the affinity methods used to deplete high abundant serum proteins target native proteins under nondenaturing conditions, these methods are also likely removing those proteins or peptides bound to the target protein. Hence, an ideal fractionation/depletion method would completely remove highly abundant proteins but leave remaining those peptides and proteins bound to them. Low molecular weight human serum proteins, peptides, and other small components have been associated with pathological conditions such as cancer (11Petricoin E.F. Ardekani A.M. Hitt B.A. Levine P.J. Fusaro V.A. Steinberg S.M. Mills G.B. Simone C. Fishman D.A. Kohn E.C. Liotta L.A. Use of proteomic patterns in serum to identify ovarian cancer. Lancet. 2002; 359: 572-577Google Scholar), diabetes (12Basso D. Valerio A. Seraglia R. Mazza S. Piva M.G. Greco E. Fogar P. Gallo N. Pedrazzoli S. Tiengo A. Plebani M. Putative pancreatic 2002; Scholar), and and R.B. M. A protein that of cancer and other Scholar), and likely the of the cell or A surface-enhanced laser/desorption ionization mass with to identify proteomic patterns in serum identified species with molecular that to the of from disease within the (11Petricoin E.F. Ardekani A.M. Hitt B.A. Levine P.J. Fusaro V.A. Steinberg S.M. Mills G.B. Simone C. Fishman D.A. Kohn E.C. Liotta L.A. Use of proteomic patterns in serum to identify ovarian cancer. Lancet. 2002; 359: 572-577Google Scholar). that the LMW serum proteome may an archive of histological information and biomarkers for disease detection. The identification of these low abundant protein however, is by the presence of the more abundant proteins in serum. A method for the removal of high molecular weight species from serum the of LMW components has been developed in this This method centrifugal ultrafiltration solvent conditions that serve to protein-protein that LMW components that may be bound to larger species are and are to the molecular weight The LMW serum proteome was digested with trypsin, and the peptide was fractionated by strong cation exchange of these was analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass analysis of the spectra resulted in the identification of human serum proteins, and peptides from human serum albumin identified in of the The large number of proteins identified demonstrates the efficacy of enrichment method with fractionation and analysis for the characterization of the LMW serum proteome. This method a and to for those components that have been to be in the early diagnosis of such as ovarian cancer. centrifugal with a of from human serum was from the of and and from was from was from and was from and high liquid and was from reversed-phase from The centrifugal and used to the of human serum was by the of of and a centrifugal The sample was in a of the serum the The was to and in of An of the was analyzed by a or a The of the LMW was by for to the sample was with for by 10 for was to the and LMW a ratio of by While was to the the for a high that a of peptides by which to characterize proteins within the LMW serum fraction S. A method to for proteome analyses. 2002; Scholar). The was by with to a concentration of and a reversed-phase as the The from the was to and in of and the and or most of the protein in the to the LMW A of of protein was from the serum sample that been in the presence of that the LMW fraction of the serum protein an of could be expected with 10 of serum. the of a of analysis of human serum in and nondenaturing Serum was with or and to centrifugal of the serum to and with and molecular weight and serum and serum affinity with 10 with and with The used was The with the affinity is however, protein with an (i.e. may bind to an of serum, serum nondenaturing conditions (i.e. and serum conditions (i.e. in by a in The serum to the a The for with The with by a The was removed and the of a in to of the and in the mass and mass spectra the in ionization and a mass of The mass was the peptide mass The digested LMW serum sample that been in the presence of was fractionated chromatography an coupled on-line to an The separation was a A a of The was with solvent A of the sample was the and with solvent over the A for and and for to and in of prior to analysis. reversed-phase was an coupled on-line to an ion-trap mass of sample 10 that in with of the was for with solvent A in and the peptides a of solvent in to solvent in a of The was in a in which was by the most abundant peptide molecular for collision-induced a of The of the and electrospray and respectively. spectra from the the human proteomic a An approach to tandem mass of peptides with in a protein J. Scholar). For a peptide to be a identification it to the and proteolytic in J.N. Varnum S.M. Auberry K.J. Moore R.J. Angell N.H. Smith R.D. Springer D.L. Pounds J.G. Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry. Mol. Cell. Proteomics. 2002; 1: 947-955Google Scholar). A of was for an identification to be of the spectra the human removing of the proteins from the protein over within this with human that was identified as from a protein was removed as a because the serum sample used in this is known to be and The of these in the has the as the from other J.N. Varnum S.M. Auberry K.J. Moore R.J. Angell N.H. Smith R.D. Springer D.L. Pounds J.G. Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry. Mol. Cell. Proteomics. 2002; 1: 947-955Google Scholar) or other such as J. of multidimensional chromatography coupled with tandem mass for protein the proteome. J. Res. 2003; 2: Scholar). the tandem spectra that identified human proteins a single peptide for and the presence of or used in the analysis of the spectra for peptides within the LMW serum only in a such as serum thousands of proteins and peptides that are present in a large dynamic concentration range from the highly abundant proteins as to the extremely low abundant proteins as the peptide P. Y. A. D. P. in with and 2003; Scholar). A major to the analytical of the serum proteome is that a single albumin, of the protein content only 10 proteins of the protein content of serum. the remaining proteins up of this remaining only of the protein content of serum is made up of proteins that are to be in low abundance and of interest in proteomic in of potential biomarkers. While the depletion of proteins to be highly abundant a removing albumin would have a in the to characterize the serum proteome by and methods have been used to remove albumin (8Sato A.K. Sexton D.J. Morganelli L.A. Cohen E.H. Wu Q.L. Conley G.P. Streltsova Z. Lee S.W. Devlin M. DeOliveira D.B. Enright J. Kent R.B. Wescott C.R. Ransohoff T.C. Ley A.C. Ladner R.C. Development of mammalian serum albumin affinity purification media by peptide phage display. Biotechnol. Prog. 2002; 18: 182-192Google Scholar, M. The of human serum characterization and J. Scholar, D.L. A. D.B. of the highly abundant protein albumin from human plasma the Proteomics. 2003; Scholar). LMW proteins are however, as albumin is known to as a carrier and transport protein within blood and is likely to bind many species of interest such as peptide hormones, cytokines, and (10Burtis C.A. Ashwood E.R. Tietz Fundamentals of Clinical Chemistry. 5th Ed. W. B. Saunders Company, Philadelphia, PA2001Google Scholar). have centrifugal ultrafiltration and have the highly abundant proteins such as albumin and for LMW proteins. the role of albumin as a transport protein in centrifugal ultrafiltration was conducted a to potential interactions. The serum analyzed by and A depletion of albumin was in the as in albumin could be by and While the depletion of albumin by ultrafiltration has the potential to concomitantly remove LMW proteins/peptides with albumin, it is important to the ultrafiltration under conditions that these that the conditions used potential LMW and albumin, the of not to the serum sample prior to An serum was with to which been to a concentration of a of serum was with and the was analyzed by While the presence of the to deplete albumin, it have a the enrichment of LMW proteins, as in The of and in of LMW species with the sample to which been and patterns under conditions serum was in of not This that the in of the LMW fraction is a of of the more abundant proteins a in due to the presence of A was in and of this The was and these from the digested with trypsin, and the peptides analyzed by This was identified as the of mass of the resulting serum also analyzed by D. R. for diagnostic 2003; Scholar). The spectra of the serum that in the presence and of are in The mass of the sample in the presence of many more than serum is with the however, the of the serum While this is not it may be due to as in highly abundant serum proteins that have high molecular not in analysis. This is of the protein the are with the in that the presence of in the is for enrichment of LMW serum proteins. methods are widely used to characterize protein proteolytic and the of peptide separation by chromatography coupled on-line with tandem are for the detection of small peptides and proteins. multidimensional fractionation the complexity of the being analyzed resulting in increased and the dynamic range of the to the complexity of the individual that analyzed by peptides from human serum in a first a A and to that analyzed by The spectra from the the human proteomic the in of the spectra that resulted in are in of the spectra to peptides that the and This analysis resulted in the identification of proteins in the human serum LMW proteome. The identified proteins are in the which the identified the number of a peptide from that protein was the number of peptides identified for that as well as the peptide with the The efficacy of the ultrafiltration method to deplete albumin, for example, is by the that not a single peptide from this protein was identified in the analysis of the The proteins identified in from a range of as in For example, many proteins being present in serum, such as circulatory proteins, and transport and proteins, cytokines, and A number of proteins not associated with serum, such as proteins, and also identified the that cell may be the and J.N. Varnum S.M. Auberry K.J. Moore R.J. Angell N.H. Smith R.D. Springer D.L. Pounds J.G. Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry. Mol. Cell. Proteomics. 2002; 1: 947-955Google the of proteins identified within the LMW serum Low abundant proteins up of the human serum with the remaining being of only proteins. It is to deplete the level of abundant proteins as an essential first step in the characterization of serum by analyses. (e.g. have been used to remove abundant proteins such as albumin, however, these methods likely in the removal of LMW species bound to albumin (10Burtis C.A. Ashwood E.R. Tietz Fundamentals of Clinical Chemistry. 5th Ed. W. B. Saunders Company, Philadelphia, PA2001Google Scholar). used centrifugal ultrafiltration to deplete abundant proteins such as albumin and for LMW proteins. The method for the and removal of albumin and other highly abundant proteins the of LMW components potentially bound to high abundant proteins. An by M.S. analysis of human of centrifugal ultrafiltration to remove albumin and other high molecular weight proteins. Proteomics. 1: Scholar) that ultrafiltration to remove albumin and other high molecular weight proteins from human the a than the serum that was with albumin in the The in was highly for LMW proteins as by MS, and A of the conditions used in and that in which plasma was used potential for this the serum conditions to protein-protein for example, species to the the of conditions, as enrichment for LMW proteins is the ultrafiltration is conducted under nondenaturing solvent The not the plasma prior to the in was conducted low (i.e. conducted At this high centrifugal it is that the of the may have been high molecular weight components such as albumin to The solvent conditions used in are also to other fractionation methods such as chromatography, which could in a more defined low molecular of which fractionation method is the that the to deplete high abundant such as albumin, for LMW proteins is highly the solvent conditions While of the of this was to an fractionation method to enable the identification of components of the LMW serum the of proteins in the information reveals the presence of many proteins that molecular mass than For example, molecular mass of the of the first protein in this has a molecular mass of The identification of peptides from proteins with than is due to the high content of serum. the presence of proteins with high molecular in the LMW serum a LMW serum was by and and identified by peptides from proteins with molecular than identified and it that the LMW proteome of serum is of many proteolytic fragments from larger proteins and it be to be of proteins. The used to identify the proteins present in the LMW fraction by used to the and D. analysis of the proteome by multidimensional protein identification Biotechnol. Scholar). in the the LMW serum proteome was digested with trypsin, serum a number of resulting in a number of peptides (i.e. R. P. M. M. of the peptide fraction in human blood of human J. Scholar). in analysis a number of serum Because analysis the LMW a larger of and peptides are expected than the proteome are with those in the by also identified a large number of peptides fractionation by analysis as well as identified most of proteins a single peptide identification J.N. Varnum S.M. Auberry K.J. Moore R.J. Angell N.H. Smith R.D. Springer D.L. Pounds J.G. Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry. Mol. Cell. Proteomics. 2002; 1: 947-955Google Scholar). The most extensive characterization and of serum proteins to is that of in which proteins identified J.N. Varnum S.M. Auberry K.J. Moore R.J. Angell N.H. Smith R.D. Springer D.L. Pounds J.G. Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry. Mol. Cell. Proteomics. 2002; 1: 947-955Google Scholar). a was used to deplete immunoglobulins prior to a of proteins of the the only proteins identified in of the thus the of identification of well proteins. albumin peptides the of centrifugal ultrafiltration for high mass and high abundant protein only peptides identified as from or transferrin, other highly abundant serum proteins. It to be that in the of a of the serum its LMW the presence or of a protein might be a of The the proteins identified in this and in those conducted by Anderson (7Anderson N.L. Anderson N.G. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics. 2002; 1: 845-867Google Scholar) and J.N. Varnum S.M. Auberry K.J. Moore R.J. Angell N.H. Smith R.D. Springer D.L. Pounds J.G. Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry. Mol. Cell. Proteomics. 2002; 1: 947-955Google Scholar) is and respectively. This low of is not as are several in the sample and analysis in of the The by Anderson proteins from plasma that been identified with analysis (7Anderson N.L. Anderson N.G. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics. 2002; 1: 845-867Google Scholar). The of a separation and analysis approach to with fractionation only depletion J.N. Varnum S.M. Auberry K.J. Moore R.J. Angell N.H. Smith R.D. Springer D.L. Pounds J.G. Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry. Mol. Cell. Proteomics. 2002; 1: 947-955Google Scholar). The within to for the LMW proteome would in a protein content of the serum sample being that identified in this as well in the by Anderson (7Anderson N.L. Anderson N.G. 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